Bioinformatic analysis of endometrial miRNA expression profile at day 26–28 of pregnancy in the mare

The establishment of the fetomaternal interface depends on precisely regulated communication between the conceptus and the uterine environment. Recent evidence suggests that microRNAs (miRNAs) may play an important role in embryo-maternal dialogue. This study aimed to determine the expression profile of endometrial miRNAs during days 26–28 of equine pregnancy. Additionally, the study aimed to predict target genes for differentially expressed miRNAs (DEmiRs) and their potential role in embryo attachment, adhesion, and implantation. Using next-generation sequencing, we identified 81 DEmiRs between equine endometrium during the pre-attachment period of pregnancy (day 26–28) and endometrium during the mid-luteal phase of the estrous cycle (day 10–12). The identified DEmiRs appear to have a significant role in regulating the expression of genes that influence cell fate and properties, as well as endometrial receptivity formation. These miRNAs include eca-miR-21, eca-miR-126-3p, eca-miR-145, eca-miR-451, eca-miR-491-5p, members of the miR-200 family, and the miRNA-17-92 cluster. The target genes predicted for the identified DEmiRs are associated with ion channel activity and sphingolipid metabolism. Furthermore, it was noted that the expression of mucin 1 and leukemia inhibitory factor, genes potentially regulated by the identified DEmiRs, was up-regulated at day 26–28 of pregnancy. This suggests that miRNAs may play a role in regulating specific genes to create a favorable uterine environment that is necessary for proper attachment, adhesion, and implantation of the embryo in mares.


The miRNA expression profile in equine endometrium
The sequencing of examined samples provided from 2 435 739 to 18 697 944 reads (42 NTs) per sample.The number of short-sequence reads obtained for each RNA sample is presented in Table 1.After rejecting low-quality reads (reads length < 18 and > 30 NTs, reads without a 3′-adapter or reads containing non-determined nucleotides "N") and 3′-adapter sequences, the remaining reads (2.19-14.49 million reads per sample), were mapped to the sequences annotated as non-coding RNAs, except miRNAs, in the horse reference genome.The percentage contribution of specific ncRNA subtypes removed from the dataset is presented in Supplementary Table 1.Next, the remaining reads (1.96-10.49 million reads per sample) were mapped to the mature miRNA sequences in the miRBase database.The percentage of the aligned reads ranged from 47.9 to 65.2%, and an average of 98.9% of these reads were mapped to a unique (i.e., only one) location.The total number of mature miRNAs expressed in all examined samples ranged from 300 to 406 (Table 1), most of which were 21-23 NTs in length (88.44%), with the highest proportion being 22 NTs (48.59%), followed by 23 NTs (21.85%) and 21 NTs (18.00%;Supplementary Fig. 1, Supplementary Table 1).The PCA shows that the miRNAs identified in equine endometrium tissue during days 26-28 of pregnancy were clustered separately from those identified in endometrial tissue during the mid-luteal phase of the estrous cycle (Fig. 1).Volcano plot depicts the distribution of transcripts, including DEmiRs, identified in the examined samples (Fig. 2).

Functional classification of DEmiR target genes
To indicate the possible role of the miRNAs identified in the current study, a functional analysis of DEmiR target genes was performed.These genes were then classified into the 'molecular function' category of GO terms, where

Discussion
microRNAs have been widely reported to be involved in embryo-uterine cross-talk during the preimplantation period of pregnancy in multiple species, including primates, rodents, and pigs 20 .In this study, 81, including 48 up-regulated and 33 down-regulated DEmiRs were identified in the equine endometrium during the preattachment period of pregnancy compared to the mid-luteal phase of the estrus cycle.A total of 4460 targets were predicted for the identified DEmiRs.These targets were mainly involved in sphingolipid metabolism, as well as ligand-gated channel activity, transmitter-gated ion channel activity, and neurotransmitter receptor activity.These processes are important for creating a favorable uterine environment for proper embryo attachment, adhesion and implantation.
Sphingolipids, vital components of the cell membrane, play an important role in regulating cellular fate in reproductive processes, particularly during pregnancy 21 .Disruptions in sphingolipid metabolism can impair uterine blood vessel formation and cause early pregnancy loss in mice 22 .Furthermore, sphingolipids regulate the expression of actin-binding proteins (ERM protein family), affecting cell adhesion, which is crucial for embryouterine interaction 23,24 .Data concerning the involvement of miRNAs in regulating the expression of enzymes associated with sphingolipid biosynthetic pathways are limited.Results of the studies carried out mostly in cancer cell lines, demonstrated that miR-9, miR-29b, and miR-101 regulate the expression of enzymes involved in the biosynthesis of sphingolipids, including ceramide [25][26][27] .However, as far as we know, no study has yet demonstrated the expression of genes associated with sphingolipid metabolism and their regulation by miRNA in the mare endometrium during early pregnancy.Nevertheless, further research is required to fully investigate this matter, based on findings in various species and the results of our investigation.Ion channels are vital membrane proteins that facilitate the transfer of ions across the cell or organelle membrane, resulting in changes in membrane potential, ion gradients, pH, and second messenger signaling [28][29][30][31] .Their expression was found in the human, rodent, and porcine endometrium [28][29][30][31] .Additionally, Cl−, K+, and Na+ channels have been recently detected in the endometrium of pregnant mares 19 .In the uterus, ion channels primarily regulate the volume and composition of the electrolyte and water-based fluid.A reduction in the volume of uterine fluid leads to the closure of the uterine lumen, preventing the embryo from moving and facilitating its implantation 32 .It was demonstrated that ion channels present in the endometrium regulate endometrial epithelial cell proliferation and apoptosis, endometrial prostaglandin synthesis, and protease activity 32 .In humans and rodents, abnormal expression or activity of endometrial ion channels resulted in implantation failure 32 .Currently,  in the literature, there is a lack of information regarding the involvement of miRNA molecules in regulating the expression and activity of uterine ion channels during embryo implantation.Previous research has primarily focused on cardiovascular (miR-21, miR-29b) and nervous systems (miR-9, miR-30b, miR-92, miR-129, miR-142), as well as cancer cell lines (miR-34), as models [33][34][35] .In conclusion, miRNAs may play an important role in embryo-uterine interaction in mares by regulating the expression of genes related to ion channel activity, thus preparing a suitable uterine environment.
The results of the current study demonstrated that the expression of eca-miR-145 was reduced in the endometrium of pregnant mares.miR-145 has been shown to be a critical regulator during embryo attachment, adhesion and implantation in humans and rats [47][48][49][50] .It regulates the expression of several genes, such as IGF1R, COX-2, MMP-9, MMP-11, VEGF, HO-1, sFRP, and FSCN-1, which are recognized for their pro-angiogenic and anti-inflammatory properties, as well as their role in modulating cell adhesion and cell-cell interactions 37,[47][48][49][50] .Additionally, miR-145 targets MUC1 50 , which expression was found to be up-regulated in the current study.Mucin 1 is a transmembrane glycoprotein that provides lubrication, hydration, and protection against external pathogens in the endometrium.It is present in the luminal and glandular epithelium of various mammalian species, including horses, as well as in horse placental tissue 5 .In many mammalian species, the removal or downregulation of MUC1, which creates an anti-adhesive barrier that is crucial for proper embryo adherence Table 3. KEGG enrichment analysis of target genes predicted for differentially expressed miRNAs (p adjusted < 0.05, log2FC ≥ 1.0/log2FC ≤ − 1.0) in mare endometrium samples obtained during pre-attachment period of pregnancy (day 26-28).and implantation, occurs.However, this does not seem to be the case in horses 51 .The equine embryo strategy for adhesion to endometrial epithelium appears to differ from that of other species.Despite being 'fixed' at the base of a uterine horn from about day 16, the equine embryo remains unattached to the endometrial luminal epithelium until day 40 52 .According to Wilsher and colleagues 5 , MUC1 protein is present at the embryo-maternal interface during different stages of equine pregnancy, including the pre-attachment stage (days 20-37).They concluded that equine embryo implantation occurs regardless of the presence of MUC1.However, there are indications that, despite its well-known anti-adhesive properties, MUC1 may also facilitate cell adhesion.The structure of MUC1 oligosaccharides can change to enable binding with the trophoblast.Therefore, certain MUC1 glycoforms, such as those bearing LNF-1 or selectin ligands, may have pro-adhesive properties 53 .Hey and Aplin 54 reported  that MUC1 can bind intercellular adhesion molecules, such as SLex and Slea.However, the exact mechanisms by which this protein may influence embryo adhesion and implantation in the mare remain unclear due to conflicting reports of its pro-and anti-adhesive properties.Further research is required to determine the specific role of MUC1 during the pre-attachment period of equine pregnancy, as well as the role of miR-145, which regulates its expression.The present study found that the eca-miR-34b, eca-miR-423, eca-miR-491-5p and eca-miR-500 expression was downregulated in pregnant mares.These miRNAs were previously shown to regulate the expression of LIF [55][56][57][58] , which was found to be up-regulated in pregnant endometrium in the current study.An increase in the expression of LIF and its receptor has been observed during the peri-implantation period of pregnancy, particularly during the establishment of uterine receptivity, in various species, including horses 19,59,60 .LIF may enhance embryo attachment, adhesion and implantation through various mechanisms.LIF activates the JAK-STAT3 pathway, influencing uterine physiology, including cell fate, angiogenesis, and innate immune response 61 .Additionally, LIF promotes the expression of miR-21 via STAT3 activation, facilitating epithelial-mesenchymal transition crucial for embryo implantation 62 .LIF also stimulates the production of MMP-9 and uPA, enzymes involved in tissue remodeling and angiogenesis 63 .While LIF appears to be a shared target for multiple miRNAs during equine endometrial receptivity formation, further research is essential to deepen our comprehension of LIF and the associated miRNA regulatory pathways during embryo attachment and implantation in the mare.
In conclusion, it appears that the identified in the current study miRNAs may influence the expression of numerous genes, including those involved in the ion channel activity, sphingolipid metabolism, formation of endometrial receptivity, angiogenesis, and ECM remodeling.Our results suggest that the identified miRNAs may be important for successful embryo attachment, adhesion and implantation through the preparation of a suitable uterine environment in mares.Identifying significant miRNAs in the pregnant equine endometrium is crucial for understanding the role of these molecules during the pre-attachment period of pregnancy.This knowledge may help reduce the rate of pregnancy loss in the future.However, more detailed research is required to specify the role of individual miRNAs during early pregnancy in the mare.

Animal study and tissue collection
Endometrial tissue from six clinically healthy, normally cycling, multiparous, mixed breed mares (aged 3-6 years, weighing 500 ± 100 kg) was used in this study.The procedures were reviewed and approved by the Local Ethics Committee for Experiments on Animals in Olsztyn, Poland (approval number 51/2011).The study was carried out between April and June 2016.The mares were housed in private stables with ad libitum access to water and fed hay and cereals.The horses were considered to be healthy based on a physical examination by a veterinarian.The animals also had no reproductive tract abnormalities, which was confirmed by ultrasonography.In addition, the endometrium was of category I according to Kenney and Doig 64 as assessed by microscopic observation after hematoxylin-eosin staining.Prior to the experiment, mares received two doses of a PGF2α analog (5 mg dinoprost; Dinolytic, Zoetis, Poland) 12 days apart to synchronize estrus.Follicular development was monitored in the mares by transrectal ultrasonography (USG) using a 7.5 MHz linear probe (MyLabOne Vet Ultrasound System; ESOATE Pie Medica, Genoa, Italy) at 12-h intervals during the periovulatory period until ovulation.In addition, visible signs of estrus (i.e., vaginal mucus and standing behavior) and structural changes in the corpus luteum were assessed by USG every 2 days until day 10 (day 0 = ovulation day).Three mares were inseminated by natural mating with the same stallion on day 0 of the estrous cycle.The day after mating was identified as the first day of pregnancy.Pregnancy was determined by USG and additionally confirmed by embryo flashing/ collection from the uterus during slaughter.Uteri were collected from mares at a local abattoir on days 10-12 of the estrous cycle (control group; non-inseminated mares; n = 3) and days 26-28 of pregnancy (inseminated mares; n = 3).Endometrial samples were collected 5-10 min post-slaughter, transferred to RNAlater (Invitrogen, Carlsbad, California, USA), transported to the laboratory at 4 °C and immediately processed for RNA isolation and NGS.Animals were slaughtered for meat as part of routine breeding as slaughter animals.

miRNA isolation
For NGS, total RNA was extracted using the Direct-zol RNA MiniPrep Kit (Zymo Research, Irvine, California, USA) according to the manufacturer's protocol.RNA concentration and quality were measured using a Nan-oDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and a 2200 TapeStation instrument (Agilent, Santa Clara, USA); only samples with an A260 nm/230 nm ratio between 1.8 and 2.2 and an RNA integrity number greater than 7.5 were used.

miRNA sequencing
miRNA sequencing was performed as previously described 65 .Briefly, miRNA libraries were prepared using the NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs, Ipswich, MA, USA) according to the manufacturer's instructions.Specifically, after 3′ adapter ligation, hybridization of the reverse transcription primer and 5′ adapter ligation, reverse transcription and PCR amplification of the resulting products were performed.The PCR was performed using 12 different indexed primers containing a unique sequence of 6 NTs in length, allowing barcoding of each library and multiplexing of samples during sequencing.The next step was the size selection (Novex 6% TBE PAGE gel, [Invitrogen]) of the libraries.The quantity of the obtained libraries was then measured using a Qubit 2.0 Fluorometer (Thermo Fisher Scientific), while a 2200 TapeStation instrument (Agilent Technologies) was used to assess their size.The obtained libraries were then sequenced on a HiScanSQ sequencing instrument (Illumina, San Diego, CA, USA) according to the manufacturer's protocol.
To predict target genes for the identified DEmiRs, the GUUGle 73 , miRanda 74 , PITA 75 , rna22 76 and RNAhybrid 77 tools in the Tools4mirs server 78 were used.The 5'UTR, CDS and 3'UTR sequences of horse proteincoding genes were used as potential targets.In addition, the binding free energy for potential miRNA-mRNA target pairs was calculated using the PITA, RNAhybrid, rna22 and miRanda tools.Only miRNA-mRNA pairs predicted by at least three of the five tools used and with a binding free energy below -10.0 kcal mol −1 , were selected.
To explore the role of the revealed DEmiRs, the identified miRNA target genes were classified according to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) categories to provide an overview of their biological functions and to assign them to specific cellular pathways and molecular mechanisms.Functional analysis of the identified target genes according to the GO database was performed using the clusterProfiler (v3.16.1 79 ), DOSE (v3.14.0 80 ), biomaRt (v2.44.4 81 ) and AnnotationHub (v2.20.2 82 ) packages of the R software, with the established criteria p adjusted < 0.05.KEGG enrichment analysis was performed using clusterProfiler, DOSE and AnnotationHub packages of R software, with the established criteria: p adjusted < 0.05.

RNA extraction and RT-qPCR analysis
RT-qPCR was used to validate the NGS results and to determine the expression of genes that are potential targets of DEmiRs.The same tissue samples were used for both NGS and RT-qPCR.In detail, total RNA was extracted from endometrial tissue using a mirVana isolation kit (Invitrogen) according to the manufacturer's instructions.The concentration and quality of total RNA was determined spectrophotometrically.The A260/280 ratio was approximately 2. Total RNA was reverse transcribed using a TaqMan MicroRNA Reverse Transcription Kit (Invitrogen) with specific RT primers or a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, Massachusetts, USA) with RNaseOUT™ Recombinant Ribonuclease Inhibitor (Invitrogen) to examine changes in miRNA or mRNA expression profiles.The qPCR was performed using prepared cDNA, specific primers and probes, and TaqMan Universal PCR Master Mix II (Applied Biosystems).The qPCR conditions were set as recommended by the manufacturer: initial denaturation for 10 min at 95 °C, 45 cycles of denaturation for 15 s at 95 °C and primer annealing for 1 min at 60 °C.The qPCR data were analyzed as previously described 84 .Data are expressed as mean ± SD.Statistical analysis was performed using Student's t-test (GraphPad Prism software, version 7; GraphPad; San Diego, USA).Results were considered significantly different at p < 0.05.

Figure 2 .
Figure 2. Volcano plot presenting all miRNAs, including differentially expressed miRNAs (DEmiRs; p adjusted < 0.05, log2FC ≥ 1.0/log2FC ≤ − 1.0) identified in mare endometrium tissue.Please note that DEmiRs are represented by multicolored circles, where red color means up-regulated DEmiRs and green color depicts downregulated DEmiRs.The grey circles represent all remaining miRNAs identified in the examined samples.

Figure 6 .
Figure 6.Dot plot illustrating Gene Ontology (GO) pathway enrichment analysis of the target genes predicted for differentially expressed miRNAs (DEmiRs; p adjusted < 0.05, log2FC ≥ 1.0/log2FC ≤ − 1.0) identified in mare endometrium tissue during pre-attachment period of pregnancy (day 26-28).The size of dots depends on number of target genes assigned to particular processes, while the dot color depends on pathway enrichment significance.

Figure 7 .
Figure 7. KEGG analysis of target genes predicted for differentially expressed miRNAs (DEmiRs; p adjusted < 0.05, log2FC ≥ 1.0/log2FC ≤ − 1.0) identified in mare endometrium tissue during pre-attachment period of pregnancy (day 26-28), and assigned to the sphingolipid metabolism pathway.The target genes predicted for DEmiRs are marked in green.

Figure 8 .
Figure 8. Real-time validation of the selected differentially expressed miRNAs (DEmiRs; a eca-miR-19a; b eca-miR-21) identified in mare endometrium tissue during preimplantation period of pregnancy by RNA-Seq.The validation was performed using the same RNA samples as used in the NGS.Data were expressed as mean ± SD.Statistical analysis was performed using Student's t-test.Different superscripts designate statistical differences (p < 0.05).MID: mare endometrium samples obtained during mid-luteal phase of estrus cycle; PREG: mare endometrium samples obtained during pre-attachment period of pregnancy (day 26-28).

Figure 9 .
Figure 9. Real-time determination of expression of (a) MUC1 and (b) LIF, genes potentially regulated by identified in mare endometrium tissue during pre-attachment period of pregnancy (Day 26-28) DEmiRs.Data were expressed as mean ± SD.Statistical analysis was performed using Student's t-test.Different superscripts designate statistical differences (p < 0.05).MID: mare endometrium samples obtained during mid-luteal phase of estrus cycle (day 10-12); PREG: mare endometrium samples obtained during pre-attachment period of pregnancy (day 26-28).